Westby, Michael (1991) Studies on the targeting and processing of proricin. PhD thesis, University of Warwick.

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Abstract

The targeting and processing of proricin, a precursor form of the highly cytotoxic castor bean lectin, ricin, was studied in this thesis. Targeting signals are present on plant seed storage protein precursors which first direct them into the ER-lumen (signal peptides) and then sort them to the protein bodies. Computer analysis of the 35aa residue N-terminal presequence of preproricin identified two residues as being likely sites of signal peptidase cleavage. Using site-directed mutagenesis, chimeric genes were constructed between the N-terminal region of preproricin and npt2. Expression of one such chimera in rabbit reticulocyte lysate (containing microsomal membranes) showed that the first 22aa N-terminal residues of preproricin function as a signal peptide. The ricin/npt2 chimeras were then transiently expressed in tobacco protoplasts in order to determine whether the 35aa preproricin presequence contained any vacuolar targeting information. Evidence is presented which suggests that this region is not responsible for targeting proricin to the protein bodies.

Recombinant proricin was synthesised by injection of proricin transcripts into Xenopus oocytes. Proricin synthesised in this way possessed lectin activity but did not depurinate mammalian ribosomes, indicating that the A chain domain has no RNA N-glycosidase activity when in the context of the precursor. In order to demonstrate the conversion of an inactive enzyme precursor to an active mature enzyme (the conversion of proricin to ricin), an assay was designed to purify the proricin processing enzyme from the protein bodies of dry castor bean seeds. Radiolabelled proricin was made in vitro by expression of proricin transcripts in wheat germ lysate. This was then used as a substrate to identify processing activity present in protein body extracts from dry castor bean seeds. Processing of the 63kD labelled proricin polypeptide to products of 30kD was observed by SDS-PAGE analysis and fluorography of assay samples. However, the levels of enzyme activity in the dried seed were too low to enable purification of the proricin processing enzyme. Therefore, an alternative strategy was designed to enable in vitro proteolytic cleavage of proricin. Site-directed mutagenesis was used to design proricin linkers encoding a recognition sequence for either Factor Xa or thrombin. Proricin clones containing these mutant linkers were expressed in E.coli. wheat germ lysate and a Xenopus egg-cell free extract"! When expressed in Xenopus egg cell-free extract, the proricin mutants were shown to have lectin activity, indicating that the B chain domain was biologically active. Evidence for Factor Xa-specific cleavage of proricin with a mutated linker is presented.

Item Type:	Thesis or Dissertation (PhD)
Subjects:	Q Science > QP Physiology
Library of Congress Subject Headings (LCSH):	Lectins, Ricin, Peptidase, Wheat germ
Official Date:	1991
Dates:	Date Event 1991 UNSPECIFIED
Institution:	University of Warwick
Theses Department:	Department of Biological Sciences
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Roberts, L. M. (Lynne M.)
Sponsors:	Science and Engineering Research Council (Great Britain)
Extent:	xviii, 246, [15] leaves : charts, illustrations
Language:	eng

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